A Comparative Study on the Lysosomal Cation Channel TMEM175 Using Automated Whole-Cell Patch-Clamp, Lysosomal...

Area of Science:

• Biophysics
• Neuroscience
• Molecular Biology

Background:

• The lysosomal cation channel TMEM175 is implicated in Parkinson's disease pathogenesis.
• Current electrophysiological methods like lysosomal patch-clamp (LPC) are not suitable for high-throughput screening (HTS).

Purpose of the Study:

• To develop and validate a high-throughput electrophysiology method for TMEM175.
• To characterize TMEM175 ion transport properties and pH dependence.
• To establish HTS assays for TMEM175 drug screening.

Main Methods:

• Solid supported membrane-based electrophysiology (SSME) was employed for direct lysosomal access and HTS.
• Ion translocation through TMEM175 was measured using concentration gradients and resting potential.
• pH dependence of TMEM175 conductivity was assessed.
• Tool compounds were used to evaluate SSME and automated patch-clamp (APC) assays.

Main Results:

• SSME successfully recorded concentration-dependent K+ responses, revealing two conducting states.
• Measured H+ fluxes confirmed TMEM175 proton permeability (P_H/P_K = 48,500), consistent with patch-clamp studies.
• TMEM175 conductivity was inhibited by decreased cytosolic pH but unaffected by lysosomal pH or gradients.
• SSME and APC assays were used to evaluate known TMEM175 inhibitors and enhancers, with EC50 data generated for novel enhancers.

Conclusions:

• SSME is a validated HTS-compatible method for studying TMEM175.
• TMEM175 exhibits distinct ion conducting states and sensitivity to cytosolic pH.
• This SSME approach facilitates drug discovery for Parkinson's disease by enabling high-throughput screening of TMEM175 modulators.